A bispecific T cell engager recruits both type 1 NKT and Vγ9Vδ2-T cells for the treatment of CD1d-expressing hematological malignancies

Summary Bispecific T cell engagers (bsTCEs) hold great promise for cancer treatment but face challenges due to the induction of cytokine release syndrome (CRS), on-target off-tumor toxicity, and the engagement of immunosuppressive regulatory T cells that limit efficacy. The development of Vγ9Vδ2-T cell engagers may overcome these challenges by combining high therapeutic efficacy with limited toxicity. By linking a CD1d-specific single-domain antibody (VHH) to a Vδ2-TCR-specific VHH, we create a bsTCE with trispecific properties, which engages not only Vγ9Vδ2-T cells but also type 1 NKT cells to CD1d+ tumors and triggers robust proinflammatory cytokine production, effector cell expansion, and target cell lysis in vitro. We show that CD1d is expressed by the majority of patient MM, (myelo)monocytic AML, and CLL cells and that the bsTCE triggers type 1 NKT and Vγ9Vδ2-T cell-mediated antitumor activity against these patient tumor cells and improves survival in in vivo AML, MM, and T-ALL mouse models. Evaluation of a surrogate CD1d-γδ bsTCE in NHPs shows Vγ9Vδ2-T cell engagement and excellent tolerability. Based on these results, CD1d-Vδ2 bsTCE (LAVA-051) is now evaluated in a phase 1/2a study in patients with therapy refractory CLL, MM, or AML.


INTRODUCTION
T cell-engaging therapies are promising approaches in an expanding number of malignancies. 1 Bispecific T cell engagers (bsTCEs) offer ''off-the-shelf'' immunotherapies that induce signaling most commonly via the CD3-T cell receptor (TCR) complex upon binding to a second target protein on tumor cells, thereby triggering T cell-mediated tumor lysis. 1,2 In particular in malignancies of B cell origin, impressive clinical results have been obtained with CD3-targeting bsTCEs (e.g., blinatumomab), but toxicities such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) often occur, and broadening of the use of bsTCE, specifically to solid tumors, has been challenging. [1][2][3][4] Indeed, to mitigate toxicity, priming doses and corticosteroid pre-medication, which might negatively affect clinical outcome, are often used. 1,5 Moreover, concomitant engagement of regulatory T cells (Tregs) by CD3-targeting bsTCEs is known to negatively affect clinical outcome and can even interfere with antitumor activity in patients treated with blinatumomab. 6,7 Engaging innate-like T cell subsets with inherent antitumor activity, such as type 1 natural killer T (NKT) and Vg9Vd2-T cells, could combine high therapeutic efficacy with a reduced risk of CRS and off-tumor toxicity.
Vg9Vd2-T cells represent a sizable (1%-10% of T cells) and homogeneous immune effector T cell population that responds to intracellular accumulation of phosphoantigens (pAgs) by sensing conformational changes in the butyrophilin (BTN)2A1-BTN3A1 complex. 8 Endogenous pAgs are metabolites of the mevalonate pathway and frequently accumulate in malignant cells as a result of metabolic dysregulation, 9 which sensitizes these cells for Vg9Vd2-T cell-mediated lysis. 10,11 Upon stimulation, Vg9Vd2-T cells rapidly produce T helper (T h ) 1-type cytokines, exert a direct cytotoxic effect via perforin/granzyme B and Fas ligand, and can (cross-)present antigens (Ags) in a CD1d(sulfatide tetramer) + CD1d-Vδ2 bsTCE 10   professional co-stimulatory context. [10][11][12] Both peripheral blood and tumor-infiltrating Vg9Vd2-T cell content positively correlate with clinical outcome in several malignancies. 10,11,13,14 While therapies using either pAg-based approaches alone or in combination with low-dose interleukin (IL)-2 and/or adoptive transfer of ex vivo expanded Vg9Vd2-T cells were safe and induced objective antitumor responses in several patients, the overall induction of clinical responses was modest. 11,15 One can envision that tumor-targeted activation of Vg9Vd2-T cells, an aspect that was lacking in previous studies, could significantly improve the consistency and robustness of Vg9Vd2-T cell-directed therapies. Indeed, we and others have shown successful engagement of Vg9Vd2-T cells via a bsTCE that crosslinks an epitope on either the TCR Vg9 or Vd2 chain with an epitope on a tumor-associated Ag, demonstrating antitumor activity in multiple preclinical models. [16][17][18][19][20] Type 1 NKT cells, which express a semi-invariant TCR and respond to both self and foreign (glyco)lipid Ags presented in the context of the non-polymorphic major histocompatibility complex (MHC) class I-like molecule CD1d, have well-documented antitumor properties. 10,21,22 Upon activation, this relatively rare ($0.1% of total T cells) but powerful T cell subset has multimodal activity via bidirectional crosstalk with Ag-presenting cells (e.g., B cells, dendritic cells [DCs]) inducing mixed T h 1/T h 2-type cytokines triggering downstream effector cell activation, and direct lysis of CD1d + tumor cells, tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs) via perforin/granzyme B and Fas ligand cytotoxic pathways. 10,21,22 While therapies targeting the CD1d-type 1 NKT cell axis using glycolipid Ag-based approaches and/or adoptive transfer of type 1 NKT cells were proven safe and could induce objective antitumor responses, the overall response rate was insufficient in patients. 10,21,22 Recently, we reported on the identification of a camelidderived single-domain antibody (VHH1D12) that triggers robust type 1 NKT cell activation. By using a functional and structural approach, we showed that VHH1D12 simultaneously contacts CD1d and the type 1 NKT TCR, thereby stabilizing this interaction through intrinsic bispecificity, which translates directly into antitumor activity and has the potential to overcome at least some of the clinically encountered limitations of glycolipid Agbased therapeutic approaches. Indeed, a superior effect of VHH1D12 over glycolipid Ag on type 1 NKT cell-mediated antitumor activity was noted using patient-derived tumor samples. 23 Here, we generated a CD1d-Vd2 bsTCE that can trigger robust activation of both type 1 NKT and Vg9Vd2-T cells toward CD1d + tumor cells, resulting in strong activity in in vivo models and ex vivo against patient multiple myeloma (MM), monocytic acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CLL) cells. Exploratory toxicology studies with a fully crossreactive (surrogate) CD1d-gd bsTCE in non-human primates (NHPs) demonstrated good tolerability, no clinical, chemistry, or hematology abnormalities or organ toxicity, and only low levels of IL-6.

RESULTS
Dual type 1 NKT cell and Vg9Vd2-T cell activation and antitumor activity by CD1d-Vd2 bsTCE The CD1d-Vd2 bsTCE (molecular weight [MW] $27 kDa; Table S1), generated by fusing the CD1d-specific VHH1D12 with the Vd2-TCR-specific VHH5C8, was designed to engage and conditionally activate both type 1 NKT and Vg9Vd2-T cells upon binding to CD1d. Binding experiments showed that the specificity and half-maximal effective concentration (EC 50 ) of the CD1d-Vd2 bsTCE for CD1d and Vg9Vd2-TCR was in the low-nanomolar range and equivalent to that of the individual monospecific VHHs (Figures 1A, 1B, and S1A). The CD1d-Vd2 bsTCE triggered robust upregulation of the degranulation marker CD107a on both type 1 NKT and Vg9Vd2-T cells in co-culture with multiple CD1d + (but not CD1d À ) tumor cell lines with lownanomolar and low-picomolar EC 50s , respectively (Figures 1C-1G). Equally low EC 50s were observed in co-cultures of CD1d + tumor cells and mixed (1:1) type 1 NKT/Vg9Vd2-T cells (Figures S1B-S1D). Finally, the ability of VHH1D12 to activate type 1 NKT cells ( Figure S1E) and to block diverse NKT cells (which interact with a different epitope on CD1d, adopt an alternative docking mode over CD1d, and include pro-tumor sulfatide-reactive type 2 NKT cells 22 ) was preserved. The capacity of the CD1d-Vd2 bsTCE to abrogate binding and reactivity of diverse NKT cells was shown using the sulfatide-restricted NKT cell line JRT3.DP10.7 and additionally confirmed using healthy donor peripheral blood mononuclear cell (PBMC) fractions that were enriched for sulfatide-reactive diverse NKT cells (Figures 1H, S1F, and S1G).
Cell Reports Medicine 4, 100961, March 21, 2023 3 Article ll OPEN ACCESS CD1d-Vd2 bsTCE triggered near-complete lysis of AML cell lines in co-cultures with type 1 NKT, Vg9Vd2-T cells, or a mixture thereof (E:T ratio 1:2) ( Figures 2B and S2C). Similar cytotoxic activity was observed in co-cultures with a T cell acute lymphoblastic leukemia (T-ALL) cell line, with an EC 50 in the sub-nanomolar range for type 1 NKT and in the low-picomolar range for Vg9Vd2-T cells and a mixture (1:1) of type 1 NKT/Vg9Vd2-T cells ( Figure 2C). Moreover, in 7-day co-cultures of CD1d + tumor cell lines and type 1 NKT and/or Vg9Vd2-T cells, CD1d-Vd2 bsTCE mediated antitumor activity at a low E:T ratio of 1:10, which was variably accompanied by expansion of type 1 NKT and/or Vg9Vd2-T cells ( Figures 2D, 2E, S2D, and S2E).
Importantly, in 7-day co-cultures of healthy donor-derived PBMCs (i.e., non-enriched, non-preactivated type 1 NKT and Vg9Vd2-T cells) and tumor cell lines, AML growth was controlled and MM growth further reduced in the presence of the CD1d-Vd2 bsTCE even at low to very low E:T ratios (Figure 3A). Particularly in the presence of MM cells, CD1d-Vd2 bsTCE promoted strong type 1 NKT and Vg9Vd2-T cell expansion even in the absence of exogenously added cytokines (Figures 3B and S2F-S2H). As (non-malignant) B cells and monocytes express CD1d ( Figure S2I), targeting type 1 NKT and Vg9Vd2-T cells to CD1d has a potential risk of on-target off-tumor toxicity. In co-cultures of PBMCs and MM cells, the cytotoxic response induced by CD1d-Vd2 bsTCE, which correlated with the Vg9Vd2-T cell frequency, was skewed toward malignant cells with only limited cytotoxicity toward autologous monocytes and B cells ( Figures 3C and 3D). In co-cultures of purified (untouched) Vg9Vd2-T cells with either the AML tumor cell line THP-1 or purified (untouched) monocytes (expressing similar levels of CD1d), the CD1d-Vd2 bsTCE triggered Vg9Vd2-T cells to lyse THP-1 cells but not monocytes ( Figures S2I and S2J). Collectively, these data demonstrate that CD1d-Vd2 bsTCEs trigger dual engagement of type 1 NKT and Vg9Vd2-T cells, which results in their expansion and potent and selective CD1d-restricted antitumor activity.
CD1d-Vd2 bsTCE humanization VHH have a reported low intrinsic immunogenicity risk profile due to their small size, high stability, rapid blood clearance, and high degree of sequence homology with the human immunoglobulin heavy-chain variable domain (IGHV). 24,25 Indeed, compared with the most similar human germline variable heavy-chain gene segment, CD1d VHH1D12 and Vd2 VHH5C8 had a respective 76.3% and 79.6% identity (Table S2). Nevertheless, pre-existing and infrequently occurring treatment-induced anti-drug antibodies (ADAs) have been reported, and toxicities, possibly related to ADA, have been described in clinical trials that evaluated VHH-based therapies. 24,[26][27][28][29] To further minimize immunogenicity and prevent development and/or binding of pre-existing (potentially neutralizing) ADAs, 10 humanized sequence variants of CD1d VHH1D12 and Vd2 VHH5C8 were generated and their human sequence identity and immunogenicity risk (HLA-DRB1-binding) scores were calculated (STAR Methods; Tables S1 and S2). All humanized CD1d variants had reduced CD1d binding and triggered reduced, although more variable, type 1 NKT cell degranulation ( Figures S3A and S3B). Vg9Vd2-TCR binding of all humanized Vd2 VHH variants was unaffected ( Figures S3C and S3D). Anti-Vd2 VHH variant 1 was selected as the most favorable humanized candidate (STAR Methods; Table S2) and linked to the C terminus of anti-CD1d VHH1D12 with only a Q1E substitution (this first amino acid does not interact with CD1d 23 and was substituted to increase leader peptide cleavage efficiency in the Pichia pastoris yeast strain selected for large-scale production). The humanized CD1d-Vd2 (hu-)bsTCE had an immunogenicity risk in the range of humanized antibodies. When tested against sera from healthy human donors, the hu-bsTCE showed a low incidence and level of binding to pre-existing human anti-VHH antibodies ( Figure 4A). Binding EC 50 for both CD1d and Vg9Vd2-T cells and in vitro functional activity (degranulation and tumor lysis) was identical between humanized and wild-type (WT) CD1d-Vd2 bsTCE ( Figures 4B-4D, S3E, and S3F). Similar to the CD1d-Vd2 bsTCE, the hu-bsTCE promoted expansion of type 1 NKT and Vg9Vd2-T cells, and this could be further boosted by exogenous IL-2 ( Figure 4E).
In conclusion, CD1d-Vd2 bsTCE humanization was successful and reduced the predicted immunogenicity risk without compromising functionality.
CD1d-Vd2 bsTCE triggers antitumor activity against patient-derived MM, monocytic AML, and CLL cells Next we assessed the antitumor effects of CD1d-Vd2 hu-bsTCE using patient-derived tumor samples of (myelo)monocytic and B cell lineage origin, as these generally express CD1d. 19,21,22,30,31 In bone marrow mononuclear cells (BMMCs) of patients with MM and AML and PBMCs of patients with CLL total T, type 1 NKT and Vg9Vd2-T cell frequencies and tumor cell CD1d expression were assessed. MM CD1d expression was especially high in treatment-naive patients, which is in agreement with a previous report. 30 AML CD1d expression was most pronounced on monocytic and myelomonocytic phenotypic subtypes, while CD1d was expressed on CLL cells in the majority of patients and appeared slightly higher in treated patients ( Figure 5A). Within the T cell compartment in BMMC/PBMC no statistically significant differences in type 1 NKT and Vg9Vd2-T cell frequencies were noted between MM, AML, and CLL, although the T cell frequency (of mononuclear cells) was significantly higher in BMMCs of MM patients compared with BMMCs of AML and PBMCs of CLL patients ( Figure S4A). Upon 16-h culture of patient-derived MM, monocytic AML, and CLL tumor samples with CD1d-Vd2 hu-bsTCE, clear degranulation of patient Vg9Vd2-T cells was observed ( Figure 5B). Due to the (very) low type 1 NKT cell frequency in the tumor samples, degranulation of autologous type 1 NKT cells could not be reliably determined.
In co-cultures of BMMCs or PBMCs from patients with MM, monocytic AML, or CLL and expanded healthy donor-derived  Article ll type 1 NKT, Vg9Vd2-T, or a mixture (1:1) thereof, CD1d-Vd2 hu-bsTCE induced robust type 1 NKT and/or Vg9Vd2-T cell degranulation and cytokine production; for type 1 NKT cells, both T h 1 (TNF, IFN-g, some IL-2) and T h 2 type cytokines (IL-4, IL-10); for Vg9Vd2-T cells predominantly T h 1 type cytokines (TNF, IFN-g, some IL-2) (Figures 5C-5E and S4B-S4D). Notably, levels of IL-6, a central driver of CRS symptoms secondary to chimeric-Ag receptor (CAR)-T or bsTCE treatment, 32 were consistently reduced in conditions where type 1 NKT and/or Vg9Vd2-T cells were engaged by CD1d-Vd2 hu-bsTCE. Neither stimulation of (autologous or allogeneic) type 1 NKT nor Vg9Vd2-T cells using the CD1d-Vd2 hu-bsTCE resulted in IL-17 production ( Figures 5C-5E). The CD1d-Vd2 hu-bsTCE-induced activation of type 1 NKT and/or Vg9Vd2-T cells resulted in significant lysis of patient-derived MM, AML, and CLL cells ( Figures 5C-5E). Although there was a trend between tumor CD1d expression and type 1 NKT and Vg9Vd2-T cell-mediated lysis, this did not reach statistical significance ( Figures S4E-S4G). Interestingly, and in contrast to observations using MM and AML tumor samples, the CD1d-Vd2 hu-bsTCE only triggered minimal type 1 NKT cell activation and cytolytic activity in co-cultures with CLL cells ( Figure 5E). Since we previously showed that VHH1D12-induced type 1 NKT cell activation critically depended on the presence of (weakly) agonistic glycolipid Ag in CD1d, 23 we hypothesized that CLL cells could differ from MM and AML cells in the amount and/ or type of glycolipid Ag loaded in CD1d. ER stress triggers an unfolded protein response (UPR) that has been linked to enhanced endogenous agonistic lipid Ag presentation in CD1d. 33,34 As activation of the UPR in CLL seems only partial under basal conditions, 35 we explored whether induction of ER stress in CLL cells could enhance agonistic lipid Ag loading in CD1d and thereby promote CD1d-Vd2 hu-bsTCE-mediated type 1 NKT cell reactivity. However, pre-incubation of CLL cells with thapsigargin, previously shown to induce ER stress and trigger type 1 NKT activation, 33,34 did not enhance type 1 NKT cell degranulation or CLL lysis ( Figure S4H), indicating that CLL cells might be impaired in their ability to load endogenous agonistic lipid Ag despite (induction of) ER stress. In support, pre-incubation of CLL cells with the low-affinity a-GalCer analog OCH, which loads at the cell surface and thereby bypasses the intracellular loading machinery, 36 enabled the CD1d-Vd2 hu-bsTCE to induce type 1 NKT cell degranulation, cytokine production, and cytotoxicity toward patient-derived CLL cells ( Figure 5F).
Overall, these data demonstrate that CD1d is expressed by the majority of patient-derived MM, (myelo)monocytic AML, and CLL cells and that CD1d-Vd2 hu-bsTCE strongly enhanced type 1 NKT and Vg9Vd2-T cell activation, resulting in robust cytotoxic responses toward these cells. In CLL, type 1 NKT (but not Vg9Vd2-T) reactivity depended on the presence of (exogenous) agonistic lipid Ags in CD1d.  Figure 6B). The one mouse that died likely succumbed to a small intra-peritoneal plasmacytoma causing intestinal obstruction. Persistence of type 1 NKT but not Vg9Vd2-T cells was observed in peripheral blood ( Figures S5A  and S5B). Persistence of peripheral blood Vg9Vd2-T cells appeared to be further reduced in mice treated with the CD1d-Vd2 bsTCE, possibly as a result of redistribution to (tumor) tissue, TCR downregulation, or activation-induced cell death. Nonetheless, type 1 NKT but not Vg9Vd2-T cell persistence is in agreement with previous reports and relates to cross-reactivity of human type 1 NKT for mouse CD1d and absence of similar Vg9Vd2-T cell support due to lack of BTN2A1-BTN3A1 expression in mice. 8,37,38 We next assessed the in vivo antitumor efficacy of the humanized CD1d-Vd2 bsTCE using whole PBMCs admixed with CCRF-CEM cells (ratio 1:2; Vg9Vd2-T to T-ALL ratio 1:16 and 1:47 for donor 1 and 2 respectively) in a s.c. T-ALL model in NSG mice. In the absence of PBMCs, twice-weekly i.p. treatment with CD1d-Vd2 hu-bsTCE (2 mg kg À1 ) did not inhibit tumor growth, whereas admixed PBMCs (in absence of bsTCE) slightly reduced tumor growth ( Figures 6C and S5C). A remarkable, dose-dependent inhibition of tumor growth was observed in mice with admixed PBMCs treated with the CD1d-Vd2 hu-bsTCE; tumor growth was only observed after discontinuation of bsTCE treatment ( Figure 6C and S5C). We next evaluated the effect of different dosing intervals and continuation of dosing on tumor growth in this model (donor 3; PBMC to CCRF-CEM ratio 1:1; type 1 NKT and Vg9Vd2-T to T-ALL ratio 1:455 and 1:18, respectively), using two different doses of the CD1d-Vd2 hu-bsTCE (2 mg kg À1 and 0.2 mg kg À1 ). Again, treatment with CD1d-Vd2 hu-bsTCE (2 mg kg À1 ) alone had no effect, and PBMCs alone or combined with control gp120-Vd2 bsTCE administration slightly reduced tumor growth, resulting in a minor increase in survival (median 26, 26, 31, and 29 days, respectively) ( Figure 6D). Clear tumor growth inhibition and increased survival were observed in mice receiving the combination of PBMC and either of the CD1d-Vd2 hu-bsTCE doses. Importantly, no significant differences in efficacy were observed between twice-weekly and weekly dosing, and, also, dosing once every 2 weeks resulted in a clear, albeit less pronounced, antitumor effect (median survival 54, 52, and 45 days, respectively, in mice receiving 2 mg kg À1 , and median survival 48.5, 47, and 43 days, respectively, in mice receiving 0.2 mg kg À1 ) ( Figures 6D and S5D).
Overall, these data show that CD1d-Vd2 bsTCE improves survival in multiple in vivo hematologic malignancy models using either expanded type 1 NKT and Vg9Vd2-T cells or PBMCs as effector cells and demonstrate antitumor efficacy with intermittent dosing.

Cross-reactive surrogate bsTCE in NHP induces Vg9Vd2-T cell activation and is well tolerated
To evaluate tolerability of CD1d-targeted Vg9Vd2-T cell engagement, we explored pharmacokinetic (PK) and pharmacodynamic (PD) parameters in NHP, which have a pAg-reactive Vg9Vd2-T cell population highly homologous to that of human. 39,40 The binding arms of the CD1d-Vd2 bsTCE (i.e., CD1d VHH1D12 and Vd2-TCR VHH5C8) were not cross-reactive to Macaca mulatta and/or Macaca fascicularis (origin China, Mauritius, and Vietnam) CD1d or Vg9Vd2-T cells ( Figure S6A). We therefore screened CD1d VHH1D22 (which binds a different epitope on CD1d and does not activate type 1 NKT) and a panel of Vg9-and Vd2-TCR-specific VHHs for NHP cross-reactivity. 23,41,42 CD1d VHH1D22 was cross-reactive to NHP CD1d, whereas none of the Vg9-and Vd2-TCR-specific VHHs were cross-reactive with either M. mulatta or M. fascicularis Vg9Vd2-T cells ( Figure S6A). Since no Vd2-TCR mAbs with relevant cross-reactivity have been reported, we investigated anti-Vg9-TCR mAb 7A5, for which reactivity with M. mulatta was demonstrated. 40 The 7A5 single-chain variable fragment (scFv) was linked to the C terminus of CD1d VHH1D22. Binding properties of this CD1d-Vg9 bsTCE and a control Vg9 bsTCE (containing the non-cross-reactive CD1d VHH1D12) were tested. Indeed, only the CD1d-Vg9 bsTCE specifically bound with low-nanomolar EC 50 to CD1d + NHP monocytes and nanomolar-range EC 50 to NHP Vg9Vd2-T cells (Figures 7A and S6B). Binding EC 50 of all three bsTCEs to human monocytes was equal, whereas binding EC 50 of the Vg9 bsTCEs to human Vg9Vd2-T cells was $30to 60-fold higher compared with CD1d-Vd2 bsTCE; however, this did not affect degranulation EC 50 and tumor lysis ( Figures 7A and S6C-S6E). The CD1d-Vg9 bsTCE triggered NHP Vg9Vd2-T cell degranulation with an EC 50 in the low-picomolar range ( Figure 7B), demonstrating functional equivalence for triggering monkey and human Vg9Vd2-T cell activation.
Next, CD1d-Vg9 bsTCE and the control Vg9 bsTCE were tested in an NHP single and multiple dosing (0.1, 0.3, and 1.0 mg kg À1 ) study. As expected, and related to their small size and absence of an Fc domain, a single escalating dose of either bsTCE showed a short plasma half-life (3.1-12.8 h and 3.6-27.2 h, respectively), rapid plasma clearance, and large apparent volume of distribution ( Figure S6F). Importantly, dose-dependent binding to peripheral blood Vg9-T cells was observed up to several days after injection, and was particularly prominent for the control Vg9 bsTCE, which was detectable on Vg9-T cells for up to 5 days ( Figure 7C). The shorter (detectable) duration and lower maximum binding of the CD1d-Vg9 bsTCE likely reflects differences in the size of the Ag pool (CD1d and Vg9-TCR versus Vg9-TCR alone) and differences in Vg9-T cell activation. Indeed, infusion of CD1d-Vg9 bsTCE, but not the control bsTCE, induced a transient decrease in peripheral blood Vg9-T cells with a concomitant upregulation of the activation marker CD69 (Figures 7D, 7E, and S6G).
Also, in the multiple-dosing study (seven daily doses), the binding intensity of the CD1d-Vg9 bsTCE to peripheral blood Vg9-T cells was lower compared with that observed with the control Vg9 bsTCE, although binding remained detectable throughout the study at both the 0.3-and 1.0-mg kg À1 dose levels (Figure S7A). While the CD1d-Vg9 bsTCE caused a decrease in peripheral blood and lymph node Vg9-T cell numbers during the multi-dose study, no alterations in other PBMC subsets, including B cells, T cells, and monocytes, were observed ( Figures 7F, S7B, and S7C). Importantly, infusion of CD1d-Vg9 bsTCE (but not control Vg9 bsTCE) induced a moderate dose-dependent increase in several proinflammatory cytokines (IL-2, TNF, IL-15), IL-6, and the chemokine CCL2, but only after the first administration ( Figures 7G and S7D; Table S3). Body weights were not affected by the bsTCE and clinical observations and hematological and biochemical analyses did not show bsTCE-related toxicities (Table S4). Postmortem autopsy showed only local injection site inflammation for both constructs, which likely reflects repetitive infusion procedure-related injuries. No macroscopic findings were considered related to the bsTCE and no unscheduled deaths occurred during the study. Overall, these data show that the fully NHP cross-reactive CD1d-Vg9 bsTCE has a rapid plasma clearance but prolonged on-target binding, triggering clear Vg9-T cell engagement, and was well tolerated in NHP.

DISCUSSION
CD1d is a (glyco)lipid Ag-presenting molecule frequently expressed by malignant cells of myelomonocytic and B cell lineage origin and some solid malignancies (e.g., renal cell carcinoma, medulloblastoma, and glioma), and may promote tumor growth via the presentation of (low-affinity) lipid Ags causing immunosuppressive cytokine release by type 1 NKT cells and engagement of protumoral diverse NKT cells. 10,19,21,22,30,31 We demonstrate that a bsTCE specific for both CD1d and Vd2 can induce strong CD1d-restricted activation of both Vg9Vd2-T and type 1 NKT cells, and simultaneously blocks the activation of CD1d-restricted diverse NKT cells. The CD1d-Vd2 bsTCE triggered type 1 NKT and Vg9Vd2-T cells to secrete proinflammatory cytokines, with IFN-g production being further boosted in the presence of both type 1 NKT and Vg9Vd2-T cells, possibly due to the reported stimulatory effects of type 1 NKT on Vg9Vd2-T cells. 43 Such skewing toward T h 1 cytokine responses is deemed important for downstream effector cell activation and has been linked to an improved response in several immunotherapy studies. 44,45 The frequency of type 1 NKT and Vg9Vd2-T cells in patient tumor samples was within the range of those reported in healthy human donors and, importantly, Vg9Vd2-T cells exposed to CD1d-Vd2 bsTCE in such samples could be readily activated. Of note, IL-17 production, associated with tumor-promoting activity, 46 was not observed upon CD1d-Vd2 bsTCE-mediated (autologous) type 1 NKT and Vg9Vd2-T cell activation. CD1d-Vd2 bsTCE-induced type 1 NKT and Vg9Vd2-T cell activation resulted in robust target cell lysis at low E:T ratios, indicating serial killing, and was accompanied by expansion of both type 1 NKT and Vg9Vd2-T cells even in the absence of exogenous growth-promoting cytokines. This is relevant as effector cell expansion positively relates to therapy outcome in both CAR-T cell and bsTCE therapy in patients. 47,48 Expression of CD1d on tumor cells was confirmed in the majority of MM, (myelo)monocytic AML, and CLL patients, and the CD1d-Vd2 bsTCE-mediated engagement of type 1 NKT and Vg9Vd2-T cells resulting in a T h 1 cytokine response and robust tumor cell lysis was also observed using patientderived tumor samples. Of interest was the observation that the CD1d-Vd2 bsTCE only triggered limited type 1 NKT cell activation in co-culture with patient-derived CLL cells. As CD1d expression in CLL appears to increase in more advanced stages of the disease, 19 it is tempting to speculate that CLL cells exploit the use of a distinct lipid repertoire in CD1d to circumvent type 1 NKT cell recognition, potentially in favor of recognition by pro-tumor diverse NKT cell populations. Indeed, type 1 NKT reactivity to CD1d-Vd2 bsTCE could be restored in co-cultures with CLL cells loaded with exogenous (low-affinity) lipid Ags.
Low-MW/low-affinity bsTCEs, such as blinatumomab, require continuous infusion to be effective. 49 Despite the small size of the CD1d-Vd2 bsTCE, which is below the renal threshold for first-pass clearance, 25 antitumor activity was still evident using an administration frequency of only once every 2 weeks in a s.c. T-ALL/PBMC model. Combined with the low-nanomolar (type 1 NKT) to low-picomolar (Vg9Vd2-T) EC 50s observed in in vitro experiments, and prolonged binding of the cross-reactive bsTCE to peripheral blood Vg9Vd2-T cells despite a short plasma half-life as observed in the NHP study, these data indicate an extended functional half-life and are supportive of an intermittent-dosing approach of the CD1d-Vd2 bsTCE in humans.
Common adverse events of bsTCE and CAR-T cell therapies include CRS and ICANS that are potentially life threatening and frequently mandate dose adjustment and/or immunosuppressive measures. 1,3 Both systemic activation (using glycolipid Ag-and pAg-based approaches) and adoptive transfer of type 1 NKT and Vg9Vd2-T cells were found to have a good safety profile in earlier clinical studies, 11,15,22 suggesting a low risk for CRS and/or ICANS upon CD1d-Vd2 bsTCE infusion. Nonetheless, we assessed tolerability of CD1d-gd T cell engagement in an NHP preclinical safety study. Because CD1d VHH1D22 does not share the type 1 NKT cell-stimulatory property of CD1d VHH1D12, 23 this aspect could not be studied. However, Vg9Vd2-T cells represent the substantially larger effector cell population of the two; therefore, we believe it is likely that their engagement is the dominant factor to consider. Infusion of both single and multiple doses of a cross-reactive CD1d-Vg9 bsTCE, resulting in clear Vg9Vd2-T cell engagement, was well tolerated in NHPs even at high doses of 1 mg kg À1 . Importantly, blood plasma analysis showed low levels of IL-6 release only after the first and highest dose, indicating a low risk for CRS. Furthermore, no depletion of CD1d + B cells and monocytes was observed. This is in agreement with our in vitro results showing preferential lysis of malignant cells while B cells and monocytes were relatively spared, implicating a low risk for on-target off-tumor toxicity.
Collectively, we have generated a first-in-class, off-the-shelf, humanized CD1d-Vd2 bsTCE that selectively engages both type 1 NKT and Vg9Vd2-T cells to trigger a potent antitumor response to CD1d-expressing tumor cells. Considering the expression of CD1d in CLL, MM, and AML, and the favorable tolerability profile of the surrogate engager in NHPs, the CD1d-Vd2 hu-bsTCE was termed LAVA-051 and selected for evaluation in a first-in-human clinical phase 1/2a study in patients with CLL, MM, or AML that are refractory to prior therapy (ClinicalTrials.gov identifier: NCT04887259).

Limitations of the study
Our study has several limitations. First, as mice do not have a pAg-responsive Vg9Vd2-T cell population 8 and the CD1d-specific VHH1D12 triggers activation of human but not mouse type 1 NKT cells, 23 xenograft (immunodeficient) mouse models had to be used in which downstream immune activation cannot be fully assessed, limiting their translational relevance. Second, since the CD1d-Vd2 bsTCE lacked NHP cross-reactivity, a surrogate CD1d-Vg9 bsTCE was used to explore tolerability in NHP. As the CD1d-binding arm lacked the ability to stimulate type 1 NKT cells, the impact of type 1 NKT cell activation on tolerability could not be explored. Third, while our study focused on assessing the anticancer potential of a bsTCE that selectively engages type 1 NKT and Vg9Vd2-T cells toward CD1d-expressing hematological malignances, it did not directly compare in vitro and in vivo efficacy or tolerability in NHPs with a CD3-targeting bsTCE directed against CD1d.   16-26-week-old NSG mice (Charles River) were irradiated with 2 Gy 24h prior to i.v. injection of 2.5 3 10 6 MM.1s.CD1d cells via the tail vein (day 0). On day 7, 14, and 21 PBS or a mixture of 0.5310 7 human type 1 NKT and 0.5 3 10 7 Vg9Vd2-T cells with or without CD1d-Vd2 bsTCE (100 mg per mouse) were i.v. injected. Mice were twice weekly i.p. injected with PBS or CD1d-Vd2 bsTCE (100 mg per mouse). Peripheral blood type 1 NKT (mCD45 À hCD45 + CD3 + TRAV-10 + ) and Vg9Vd2-T cell frequencies (mCD45 À hCD45 + CD3 + TRGV9 + (TRDV2 + )) were determined over time by flow cytometric bead-based cell counting. Mice were euthanized when pre-set human endpoints were reached. Blood in the one mouse, treated with type 1 NKT and Vg9Vd2-T cells plus CD1d-Vd2 bsTCE, that was found dead could not be reliably analyzed due to clotting and was excluded from the analyses. The study was terminated 90 days post tumor engraftment. All mice were maintained as described above. Animal experiments were carried out in compliance with Dutch/European ethical guidelines and approved by the Dutch Central Authority for Scientific Procedures on Animals (permission number AVD114002016402).
A s.c. T-ALL model was established via s.c. inoculation of 1 3 10 7 CCRF-CEM cells alone or mixed with 0.5-1.0310 7 (as indicated) healthy donor PBMC (day 0) in female 5-9-week old NSG mice (Vital River laboratories). Mice were i.p. injected with PBS or CD1d-Vd2 hu-bsTCE (indicated dose and frequency). Tumor volumes were measured twice a week in two dimensions using a caliper, and the tumor volume (V) calculated using the formula: V = (W 2 3L)/2, where L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L). In the dose escalating part, mice were euthanized when the tumor volume of individual mice reached >3000 mm 3 , mean tumor volume of the groups reached >2000 mm 3 or when pre-set human endpoints were reached. In the dosing interval study, mice were euthanized when the tumor volume of individual mice reached >2000 mm 3 or when pre-set human endpoints were reached. All mice were maintained as described above. The T-ALL in vivo study was performed by Crown Bioscience, China, in compliance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and approved by the Institutional Animal Care and Use Committee (IACUC) of Crown Bioscience.
NHP single and multiple dosing study PK and PD of CD1d-Vg9 bsTCE or control Vg9-bsTCE were studied in 12 female M. fascicularis, at Crown Bioscience, Beijing, China. The study was divided in two phases. Phase 1: NHP received a single dose CD1d-Vg9 bsTCE or control Vg9 bsTCE (0.1 mg kg À1 , 0.3 mg kg À1 or 1 mg kg À1 via a 30-min i.v. infusion, n = 1 animal per group) to determine PK values. Venous blood samples were collected pre-dose (day À14 and À7), 0, 0.5, 1, 2, 4, 6, and 8h, and day 1, 2, 3, 4, 5 and 6 post-infusion to determine PK values and/or flow cytometry analyses. Free plasma concentrations of the bsTCEs, measured using a free drug assay (Simoa, Quanterix) by ABL immunology (Lyon, France), were used to estimate PK parameters (PK Solver software version 2.0 55 ). Peripheral blood Vg9-T cell percentages (CD3 + TRGV9 + (RaL + )), CD69 expression, and binding of CD1d-Vg9 bsTCE or control Vg9 bsTCE to by flow cytometric counting beads. Expansion factor was calculated by dividing the cell count after co-culture by the starting cell count.
Humanization and binding of pre-existing anti-VHH antibodies In silico humanization was performed by Lonza (Lonza Biologics) via selection of human antibody germline sequences similar to monovalent VHH (CD1d VHH1D12; IMGT: IGHV3-66*01, Vd2 VHH5C8; IMGT: IGHV3-23*01), identification of VHH critical residues, and complementarity-determining region (CDR) grafting and substitution of mismatched residues between parental and acceptor framework regions, similar as described previously. 60 Sequences were analyzed for potential issues based on Ag binding, protein stability, function and sequence liabilities using a structural model. Epibase screening (Lonza) was used to screen humanized sequences for predicted T helper epitopes that may contribute to immunogenicity and to calculate an immunogenicity risk (HLA-DRB1-binding) score. For each monovalent VHH, 10 variants were generated and functionally evaluated as described above. The relative percentage of binding of the variants compared to the WT protein was calculated by dividing MF [condition] by MF [WT VHH] multiplied by 100. To generate humanized CD1d-Vd2 bsTCE (CD1d-Vd2 hu-bsTCE), CD1d VHH (clone VHH1D12.Q1E) was genetically linked by a G 4 S-linker to Vd2 TCR (clone VHH5C8.variant 1, selected based on a combination of predicted protein manufacturing characteristics, human-ness and immunogenicity risk) and expressed in P. pastoris (Validogen Gmbh, Graz, Austria). VHH1D12.Q1E (glutamic acid substitution of glutamine at position 1; a residue that was shown not to interact with CD1d nor type 1 NKT TCR) 23 was used instead of VHH1D12 WT due to reduced and inconsistent leader peptide cleavage when the Q1 containing bsTCE was produced in the P. pastoris strain (data not shown). HLA-DRB1-binding score of the monovalent binding arms of the CD1d-Vd2 bsTCE (i.e. CD1d VHH1D12 and Vd2-TCR VHH5C8) was compared to the HLA-DRB1-binding score of therapeutic human IGHV. 61 CD1d-Vd2 hu-bsTCE was functionally evaluated as described above.
The frequency of pre-existing human anti-VHH antibodies in serum was determined via an Ag binding test by Sanquin Blood Supply foundation. CD1d-Vd2 bsTCE and CD1d-Vd2 hu-bsTCE were labeled with radio-active iodine (I 125 ) and purified. I 125 -labeled bsTCE ($0.5 ng per test) was subsequently incubated with 1 mL healthy donor human serum (1:50 diluted in 0.3% PBS-BSA) and possible IgG-VHH complexes were then captured onto protein-G coated Sepharose beads. After overnight incubation and extensive washing, radioactivity of sepharose-beads was measured by a gamma counter. Specificity of the test was confirmed by inhibition of the signal by adding an excess of unlabeled bsTCE (100 ng/test, $200 times excess). Percentage of binding was calculated by dividing the number of disintegrations measured on the sepharose-bound radiolabeled bsTCE in the experimental condition by the number of disintegrations found in the total input of radiolabeled bsTCE. The cut-off point for positivity was calculated on the mean percentage binding (after removal of outliers using iterative Grubbs) plus 1.645 SD.
To be able to assess safety, PK and PD parameters in NHP, a surrogate bispecific molecule was generated using sequence information from an NHP cross-reactive mouse antibody (clone 7A5, kind gift from dr. D. Kabelitz) directed to the Vg9 chain of the TCR, coupled to the cross-reactive CD1d specific VHH1D22 (CD1d VHH1D22-scFv7A5, referred to as CD1d-Vg9 bsTCE) and produced by UPE. Bispecific molecule CD1d VHH1D12-scFv7A5 (control Vg9 bsTCE), which lacks cross-reactivity toward NHP CD1d, was generated as a control and produced by UPE. Binding of the various bispecific molecules to CD1d and Vg9Vd2-TCR was analyzed at indicated concentrations by flow cytometry as described above. For binding to monocytes and Vg9-T cells in PBMC (human or NHP), 0.25-1310 5 cells were incubated with indicated concentrations of biotinylated bsTCE for $45 min at 4 C, followed by extensive washing, incubation with Fc-block and stained with CD11b-PEcy7, streptavidin-APC and CD3-AF700. CD1d and Vg9-TCR binding was determined by streptavidin-APC MF on monocytes (CD11b ++ (CD20 À )) and Vg9-T cells (CD3 + CD11bstreptavidin-APC + ). Relative percentage of binding was calculated as described above. To evaluate induction of degranulation of Vg9-T cells, thawed human and NHP primate PBMC (0.25-1310 5 per condition) were incubated with rhIL-2 (50 IU/mL) for 24h to recover followed by incubation with or without CD1d-Vd2 bsTCE, CD1d-Vg9 bsTCE or control Vg9 bsTCE (concentration range) for an additional 24h in the presence of PE-labelled anti-CD107a, after which Vg9-T cells (CD3 + CD11b À TRGV9 + ) were analyzed for CD107a expression.

QUANTIFICATION AND STATISTICAL ANALYSIS
Sample size for the MM in vivo experiments was determined by using a two-sided, two-sample equal-variance t-test (assuming a population mean difference of 0.25 with an s.d. of 0.15 and with a significance level (a) of 0.050 with >80% power) and previous experience. Sample size for the AML and T-ALL in vitro experiments was estimated based on previous experience with these tumor cell lines. Mice were randomly assigned to treatment groups. No statistical method was used to predetermine sample size for the in vitro experiments. For in vitro experiments each n represents an independent experiment with, when applicable, primary type 1 NKT and Vg9Vd2-T cell lines, and PBMC obtained from individual healthy donors. For experiments with patient MM and AML BMMC and CLL PBMC each n represents an individual patient. For in vivo experiments each n represents an individual animal. No data were excluded from the analyses. The experiments were not randomized and the investigators were not blinded to allocation during the experiments and outcome assessment.
For data with one variable and two groups, a two-tailed paired/unpaired t-test was used to calculate the p value. For data with one variable and multiple groups, a one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test to calculate the multiplicity-adjusted p value was used. For data with two variables, a two-way ANOVA with either Tukey's or Sídá k's multiple comparisons test to calculate the multiplicity-adjusted p value was used. Dose-response curves, half maximal effective concentration (EC 50 ) was calculated using non-linear regression (agonist versus normalized response for the binding experiments and agonist versus response for the remaining experiments). To determine the relationship between CD1d-expression and cell lysis, linear regression was used to fit the line through the data points, determine goodness of fit (R2) and calculate the p value. Survival data was analyzed with Kaplan-Meier survival curves and the log rank test (two-tailed p values) was used to calculate statistically significant differences. Statistical details can be found in the figure legend. Statistical analysis was performed using Prism v.9.1.0 (GraphPad Software).